Protein Import and Export

The best characterized nucleocytoplasmic transport pathway is the classical nuclear import of proteins (Fig. 1). In a first step, importin-a proteins, which act as adaptor molecules, interact with protein cargos via targeting sequences called nuclear localization signals (NLSs). Classical NLSs consist of either one (monopartite) or two (bipartite) short stretches of basic amino acids (for a review see Lange et al. 2007). Molecular recognition of NLSs is crucial for the formation of the import complex and is mediated via specific sites on importin-a that are located within the armadillo repeat region of the protein (Conti et al. 1998; Fontes et al. 2000). Importin-a links the cargo to the P-karyopherin importin-P, which mediates interaction of the trimeric complex with the NPC and translocates the cargo into the nucleus. Within the nucleus, importin-P then binds to RanGTP, inducing a confor-mational change that results in the release of the cargo. Importin-P complexed with RanGTP is recycled to the cytoplasm, whereas importin-a is exported complexed with the P-karyopherin CAS and RanGTP. Finally, cytoplasmic RanGAP (Ran GTPase-activating protein) stimulates the Ran GTPase, which generates RanGDP and thus releases the importins within the cytoplasm for additional import cycles (reviewed in Stewart 2007).

Fig. 1 Classical cellular pathways of nuclear protein import and export. Nuclear protein import: in the cytoplasm, cargo, containing a nuclear localization signal (NLS) , is bound by the het-erodimeric import receptor, importin-a/importin-P; importin-a directly binds to the NLS-containing cargo and importin-p mediates interactions with the nuclear pore complex (NPC) during translocation. Within the nucleus, RanGTP binding causes a conformational change of importin-p, resulting in the release of the cargo. Nuclear protein export: in the nucleus, cargo, containing a leucine-rich nuclear export signal (NES) , interacts with the RanGTP complexed export factor CRM1/exportin1, which directly interacts with components of the NPC. Hydrolysis of RanGTP to RanGDP in the cytoplasm induces the release of the cargo. A gradient of RanGTP across the nuclear envelope, resulting from the activity of the chromatin-associated nucleotide exchange factor RCC1 and the cytoplasmic GTPase-activating protein RanGAP, is considered the major driving force for nuclear protein transport in both directions

Fig. 1 Classical cellular pathways of nuclear protein import and export. Nuclear protein import: in the cytoplasm, cargo, containing a nuclear localization signal (NLS) , is bound by the het-erodimeric import receptor, importin-a/importin-P; importin-a directly binds to the NLS-containing cargo and importin-p mediates interactions with the nuclear pore complex (NPC) during translocation. Within the nucleus, RanGTP binding causes a conformational change of importin-p, resulting in the release of the cargo. Nuclear protein export: in the nucleus, cargo, containing a leucine-rich nuclear export signal (NES) , interacts with the RanGTP complexed export factor CRM1/exportin1, which directly interacts with components of the NPC. Hydrolysis of RanGTP to RanGDP in the cytoplasm induces the release of the cargo. A gradient of RanGTP across the nuclear envelope, resulting from the activity of the chromatin-associated nucleotide exchange factor RCC1 and the cytoplasmic GTPase-activating protein RanGAP, is considered the major driving force for nuclear protein transport in both directions

Although the classical nuclear import pathway using importin-a as an adaptor is believed to account for the majority of nuclear protein import, several alternative pathways exist. For instance, hnRNPA1 contains a different type of NLS that is rich in aromatic residues and glycine (called M9 sequence), which binds directly to the P-karyopherin transporting resulting in docking of the complex at the NPC (Pollard et al. 1996). Also, several viral (e.g., Rex of HTLV-1and Rev of HIV-1) and cellular proteins (e.g., c-fos) are able to bind directly to various members of the importin-P family (importin-p, transportin, importin5, impor-tin7) via arginine-rich sequences without the need for an additional adapter protein (Palmeri and Malim 1999; Henderson and Percipalle 1997; Arnold et al. 2006a, 2006b).

)nterestingly, the M9 sequence not only acts as an NLS but also mediates nuclear export, thus constituting a bidirectional signal for nucleocytoplasmic shuttling (Michael et al. 1995). Nuclear export of proteins, however, is in most cases mediated via a signal sequence that is clearly distinct from the NLS. The best characterized nuclear export signal (NES) is the small, hydrophobic, leucine-rich NES, which was identified initially in the HIV-1 Rev protein and the cellular kinase PKI (Fischer et al. 1995; Wen et al. 1995). Functionally related export sequences resembling a leucine-rich NES have been detected since then in many cellular and viral proteins of diverse functions with the capacity to shuttle between the nucleus and the cytoplasm (La Cour et al. 2003). The direct interaction with the importin-P-related export factor CRM1 (exportin1) is essential for the export of proteins containing a leucine-rich NES (Fornerod et al. 1997) (Fig. 1). This interaction can be inhibited specifically by the antibiotic leptomycin B (LMB), resulting in a block of the nuclear export of proteins with a leucine-rich NES (Kudo et al. 1999). CRM1 binds cooperatively to RanGTP and its export cargo, leading to the formation of a trimeric transport complex in the nucleus. After translocation of this complex through the NPC, the cytoplasmic RanGTP-binding protein RanBP1 in concert with RanGAP dissociates the export complex (for a review see Hutten and Kehlenbach 2007).

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